Backbone cyclic insulin

Andersen, Asser S.; Palmqvist, Eva; Bang, Susanne; Shaw, Andrew; Hubálek, František; Ribel, Ulla; Høeg-Jensen, Thomas

doi:10.1002/psc.1264

Cited by 13 publications

(10 citation statements)

References 27 publications

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“…S3B) and yields preparations that are more resistant to thermal denaturation. Cyclic proteins are also resistant to exoproteolytic attack (25,26), a feature that may enhance utility of any therapeutic proteins exposed to exoproteases, for example upon receptor mediated internalization. In addition, cyclization of proteins and peptides has been shown to improve potency, stability, and oral bioavailability (27)(28)(29)(30).…”

Section: Discussionmentioning

confidence: 99%

Sortase-catalyzed transformations that improve the properties of cytokines

Popp

Dougan

Chuang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

176

150

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Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortasemediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.M any clinically relevant cytokines share a four-helix bundle structure, typified by IFNα2, Granulocyte colony-stimulating factor 3 (GCSF-3), Erythropoietin (EPO), IL-2, IL-4, IL-7, IL-9, and IL-15. Cocrystal structures of cytokines with receptor fragments and biochemical studies that map residues critical for interaction of a cytokine with its receptor show that the receptor contacts the sides of the helical bundles (1). This mode of interaction positions the N and C termini of the cytokine away from the receptor.Polyethylene glycol chains attached to therapeutically important proteins increase circulatory half-life, reduce clearance by kidney filtration, reduce proteolysis, and reduce the generation of neutralizing antibodies (2-4). The attachment of PEG commonly employs standard chemistries that target reactive amino acid side chains (e.g., cysteine and lysine). This strategy often generates a heterogeneous mixture in which multiple amino acids in the target are modified with a PEG chain, necessitating cumbersome separations and characterization (5). Such PEGylated molecules often show decreased biological activity, likely due to attachment of a PEG chain to a residue important for interaction with the receptor (6)-this problem may be overcome by sitespecific PEGylation. Although engineering of a carefully placed unpaired cysteine residue allows site-specific PEGylation (7, 8), this method must be tailored to the specific protein target.Sortase A from Staphylcoccus aureus (SrtA Staph ) is a thiolcontaining transpeptidase that recognizes an LPXTG motif in multiple structurally unrelated substrates (9). SrtA Staph cleaves the peptide bond between the threonine and glycine residues with concomitant formation of a thioacyl enzyme intermediate that involves the catalytic cysteine and the substrate threonine. This acyl-enzyme is resolved by nucleophilic attack by the N terminus of an oligo...

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Section: Discussionmentioning

confidence: 99%

Sortase-catalyzed transformations that improve the properties of cytokines

Popp

Dougan

Chuang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

176

150

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“…The mutations that appeared in the inhibitor included K→ W, G → W, K → Y and E → Y mutations with seven of the eight mutations occurred on the exposed face of a single hairpin of the B1 domain 60 . With this as an inspiration, we examined mutants of the KKLTVS-I p GK-KITVSA hairpin sequence ( p = d -Pro, to favour hairpin turn formation), with pairs of tyrosine and tryptophan residues introduced at a variety of positions, as potential inhibitors of amyloid formation from both human pancreatic amylin (hAM) and α-sy 13,61,62 . Several of these proved to be potent inhibitors of amyloid fibril formation in both cases even though they bore no structural resemblance to either of the amyloidogenic systems.…”

Section: Inhibitors Of α-Syn Amyloid Formationmentioning

confidence: 99%

Binding interactions of agents that alter α-synuclein aggregation

et al. 2015

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Further examination of peptides with well-folded antiparallel β strands as inhibitors of amyloid formation from α-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert α-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied β hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a d-Pro-l-Pro turn locus across strand termini. At a 2:1 peptide to α-synuclein ratio, cyclo-WW2 displays complete inhibition of β-structure formation. Trp-bearing antiparallel β-sheets held together by a disulphide bond are also potent inhibitors. 15N HSQC spectra of α-synuclein provided new mechanistic details. The time course of 15N HSQC spectral changes observed during β-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in 15N HSQC spectra of α-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41-T54 span of α-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor.

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“…71 Circular proteins have useful biochemical properties. They are resistant to aggregation, require more energy for denaturation, and, since they lack exposed termini, are resistant in their native form to exoprotease attack 72–76…”

Section: Piecemeal Assembly Of Proteins Protein Domains and Peptmentioning

confidence: 99%

Making and Breaking Peptide Bonds: Protein Engineering Using Sortase

2011

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Sortases are a class of bacterial enzymes that possess transpeptidase activity. It is their ability to site-specifically break a peptide bond and then reform a new bond with an incoming nucleophile that makes sortase an attractive tool for protein engineering. This technique has been adopted for a range of applications, from chemistry-based to cell biology and technology. In this Minireview we provide a brief overview of the biology of sortase enzymes and current applications in protein engineering. We identify areas that lend themselves to further innovation and that suggest new applications.

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Backbone cyclic insulin

Cited by 13 publications

References 27 publications

Sortase-catalyzed transformations that improve the properties of cytokines

Sortase-catalyzed transformations that improve the properties of cytokines

Binding interactions of agents that alter α-synuclein aggregation

Making and Breaking Peptide Bonds: Protein Engineering Using Sortase

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